It has been estimated that in the year 2000, approximately 350,000 people were living with a primary brain tumor (Davis et al. (2001) Neurooncology 3: 152–158). The expected incidence of new cases of primary brain tumors in the United States is estimated to be over 35,000 for the year 2001 (Central Brain Tumor Registry of the United States fact sheet (2001), available from CBTRUS, Chigaco, Ill.). Approximately 13,000 people die of malignant brain tumors in the United States each year, representing about 2% of all cancer deaths (Greenlee et al. (2001) Cancer J. Clin. 51: 15–36). Brain tumors are the second leading cause of cancer-related deaths in children under the age of 20 (Id.).
Glial tumors or gliomas are tumors which arise from neuroectodermal cells of the glial lineage and include glioblastomas (about 23% of primary brain tumors), astrocytomas (about 8% of primary brain tumors), anaplastic astrocytomas (about 4% of primary brain tumors), and oligodendrogliomas (about 3% of primary brain tumors). Of these, glioblastomas, which are malignant gliomas, are the most aggressive and difficult to treat (Roth & Weller (1999) Cell Mol. Life Sci. 56: 481–506; Badie & Schartner (2001) Microsci. Res. Tech 54: 106–113). Glioblastomas are the most common type of brain tumors in adults and by far the most devastating (Id.). While glioblastomas do not metastasize outside the brain, they are highly infiltrative and typically compress and destroy adjacent brain tissue and obstruct the flow of cerebrospinal fluid (CSF). Patients with glioblastomas are immunosuppressed both locally and systemically by a process which is poorly understood but which involves the secretion of TGF-β and other cytokines (Roth & Weller (1999) Cell Mol. Life Sci. 56: 481–506).
Gliomas are poorly immunogenic, and no glioma-specific antigens have been recognized (Badie & Schartner (2001) Microsci. Res. Tech 54: 106–113). While the blood-brain barrier may be disrupted in some areas affected by glioma, frequently the blood-brain barrier is intact where migrating glioma cells infiltrate into normal brain tissue (Roth & Weller (1999) Cell Mol. Life Sci. 56: 481–506). Gliomas are very heterogeneous, varying greatly not only from individual to individual, but also in the neoplastic cell populations within a tumor (Id.). Gliomas exhibit variable expression of cell surface markers. These characteristics are major impediments for therapies designed to target tumor cells.
Current treatment of glioblastomas is multimodal (Id.; Spear et al. (1998) J. Neurovirol. 4: 133–137). Surgical resection is often performed to reduce tumor load and to prevent or reduce complications associated with compression. However, it is seldom possible to completely remove the tumor due to its location and/or its infiltrative growth pattern. Radiotherapy is considered to be the most effective treatment for glioblastomas, although there are several major drawbacks to this treatment: many gliomas are resistant to irradiation-induced cytotoxicity; normal brain tissue tolerance cannot be exceeded; and radiotherapy generates late side effects (Roth & Weller (1999) Cell Mol. Life Sci. 56: 481–506). Chemotherapy is also limited in its effectiveness because of toxic effects on normal tissues, including myelosuppression and peripheral neurotoxicity (Id). While combinations of surgery, radiotherapy, and chemotherapy are currently used to treat tumors, glioblastomas are usually fatal within one to two years of onset of symptoms (Karpati et al. (1999) Curr. Opin. Mol. Ther. 1: 545–552).
One desirable alternative to these conventional therapies is more specific, genetically-based therapies to allow specific targeting of tumor cells for diagnostic and prognostic tests and for therapeutic treatments. One ligand discovery approach has been to generate monoclonal antibodies against tumor cells and screen them for specificity (see, e.g., Wikstrand et al. (1999) Cancer Metastasis Rev. 18: 451–464). While antibody-mediated targeting of tumor cells has shown clinical promise, there are several limitations on the usefulness of antibodies. Monoclonal antibody techniques are expensive, time-consuming, and dependent on the immunogenicity of targets, a property which gliomas often lack. Monoclonal antibodies may not be useful for identification and targeting of conserved cell-specific receptors due to the receptor size, structure, and/or lack of immunogenicity. Further, antibodies are relatively large, which may limit their uptake into tumor cells, and antibodies are also taken up nonspecifically by reticulendothelial cells in non-tumor tissues (Aina et al. (2002) Biopolymers 66: 184–199). Although it may be possible to improve targeting of specific cells markers by further manipulation of antibodies, antibody-based methods are currently limited in their usefulness.
Thus, there remains a need for improved therapies to treat brain tumor patients as well as for more sensitive diagnostic and prognostic techniques. Molecular profiles of neoplastic cells based on DNA, mRNA, and/or protein alterations are rapidly being developed and utilized not only to augment diagnosis but to provide new therapeutic measures. Of these profiles, protein and protein-associated markers, particularly those on the tumor cell surfaces, lend themselves most readily to targeting procedures.
Protein surface markers have been described in association with malignant glioma cells. Growth factors such as EGF, PDGF, and cytokines have been exploited because they not only bind to upregulated surface receptors, but also modulate cell proliferation and differentiation. Additionally, the structures of these factors and their receptors are known, allowing for genetic and chemical manipulations to improve binding affinity, reduce immune complications, and disrupt associated molecular pathways. Growth factor receptors that have been described in association with malignancy include: epidermal growth factor receptor (EGFR; see Kuan et al. (2000) Int. J. Cancer 88: 962–969; Wikstrand et al. (1998) J. Neurovirol. 4: 148–158); platelet derived growth factor receptor (PDGFR; see Nister et al. (1987) Cancer Res. 47: 4953–4960; Maher et al. (2001) Genes Dev. 15: 1311–1333; Westermark et al. (1995) Glia 15: 257–263). See also, Roth & Weller (1999) Cell Mol. Life Sci. 56: 481–506. Surface markers include cytokine receptors such as interleukin-4 receptor (IL-4 R; see Puri et al. (1996) Cancer Res. 56: 5631–5637; Rahaman et al. (2002) Cancer Res. 62: 1103–1109) and interleukin-13 receptor (IL-13 R; see Liu et al. (2000) Cancer Immunol. Immunother. 49: 319–324). Surface markers also include: transferrin receptor (TfR; see Li et al. (2002) Trends Pharmacol. Sci. 23: 206–209); urokinase-type plasminogen activator receptor (uPAR; see Del Rosso et al. (2002) Clin. Exp. Metastasis 19: 193–207; Kroon et al. (2000) Blood 96: 2775–2783); chloride channels (see Soroceanu et al. (1998) Cancer Res. 58: 4871–4879; Ransom et al. (2001) J. Neurosci. 21: 7674–7683) membrane-type matrix metalloproteinases (MT-MMPs; see Fillmore et al. (2001) J. Neurooncol. 53: 187–202); cell adhesion molecules such as integrins (see Goldbrunner et al. (1999) Acta Neurochir (Wien) 141: 295–305; Tonn et al. (1998) Anticancer Res. 18: 2599–2606; Paulus et al. (1996) Lab. Investigation 75: 819–826; and Laws, Jr., et al. (1993) Int. J. Dev. Neurosci. 11: 691–697); and CD44s (see Ranuncolo et al. (2002) J. Surg. Oncol. 79: 30–35; Breyer et al. (2000) J. Neurosurg. 92: 140–149). However, the presence of these receptors on normal cells, particularly during development or wound healing, makes targeting these receptors less desirable because of possible side effects on normal tissues.
The majority of markers utilized for targeting are those that are either upregulated, resulting in increased numbers of binding sites for the targeting ligand, or are rearranged molecules, allowing glioma-selective binding when compared to normal cells. For example, Debinski et al. ((2000) J. Neurooncol. 48: 103–111) showed that an increased number of binding sites for IL-13 accompanied the progression of gliomas from low to high grade. Similarly, transferrin receptors were shown to be present in significantly greater numbers on the surface of malignant glioma cell lines when compared to normal control cell lines (Wen et al. (1993) Neurosurgery 33: 878–881). In addition to cell surface markers, naturally-occurring neuroectodermal cell-specific ligands such as chlorotoxin (ClTx), a 4-kilodalton peptide from the venom of scorpions, have been used as targeting molecules for gliomas (Soroceanu et al. (1998) Cancer Res. 58: 4871–4879; Lyons et al. (2002) Glia 39: 162–173). The chlorotoxin peptide shows high-affinity, specific binding to glioma cells and may find use in therapeutic and diagnostic applications.
One of the few unique markers to tumor cells is a frequently rearranged form of EGFR known as EGFR type III variant (“EGFRvIII”), also called “de2-7 EGFR” (Johns et al. (2002) Int. J. Cancer 98: 398–408). This marker has a deletion in the extracellular domain, resulting in a tumor-specific cell surface receptor that is found on a variety of tumor cell types including malignant glioma, breast carcinoma, non-small cell lung carcinomas, and ovarian tumors (Kuan et al. (2001) Endocr. Relat. Cancer 8: 83–96).
With the exception of the rearranged EGFRvIII and IL-4-independent IL-13Rα2 (see, e.g., Nagane et al. (2001) J. Neurosurg. 95: 472–492), the surface molecules described above also are expressed by normal cells, limiting their usefulness in diagnosis or therapy. Additionally, there is frequently variation in marker expression within the same tumor mass over time with neoplastic progression or among the same type of tumors from different individuals. The multifunctional adhesion molecule involved in cell—cell and cell-matrix interactions, CD44s, was shown to be differentially expressed such that low-grade astrocytomas (9.5%) had far fewer cells with high expression of CD44s than did glioblastomas (59%), while positive CD44s staining was heterogeneous even within samples (Ranuncolo et al. (2002) J. Surg. Oncol. 79: 30–35). Such intratumoral and intertumoral heterogeneity of tumor cells indicates that no single marker will be able to provide diagnosis and/or targeting for all gliomas, but instead that an array of markers will be necessary for such purposes.
Small peptides that bind to cell surface markers also have been utilized in targeting strategies. The advantages of peptide ligands are their small size, specificity, and chemical stability, the ease with which they can be derivatized, and their general lack of binding to reticuloendothelial cells, in contrast to antibodies (Aina et al. (2002) Biopolymers 66: 184–199). Spear et al. ((2001) Cancer Gene Ther. 8: 506–511) used phage display technology to identify cell surface biorecognition molecules for brain tumors using different human and rat cell lines. However, the phage clones identified were found to have low binding specificity for glioma cells based on evaluations using ELISA. Thus, while some peptide ligands have been identified, there remains a need for additional peptide ligands which will preferentially bind to gliomas for use in diagnosis, prognosis, and therapy.